Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a null background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.
Yue
Zuojun
Z
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.
Carvalho
Ana
A
Xu
Zhenjie
Z
Yuan
Xuemei
X
Cardinale
Stefano
S
Ribeiro
Susana
S
Lai
Fan
F
Ogawa
Hiromi
H
Gudmundsdottir
Elisabet
E
Gassmann
Reto
R
Morrison
Ciaran G
CG
Ruchaud
Sandrine
S
Earnshaw
William C
WC
D020816:Amino Acid Motifs; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D064127:Aurora Kinases; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002503:Centromere / Q000187:drug effects / Q000201:enzymology; D002645:Chickens; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D048749:Cytokinesis / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013696:Temperature; D014714:Vertebrates / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D010769:Phosphothreonine; D001224:Aspartic Acid; D005047:Etoposide; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine